Qadota Hiroshi, Mayans Olga, Matsunaga Yohei, McMurry Jonathan L, Wilson Kristy J, Kwon Grace E, Stanford Rachel, Deehan Kevin, Tinley Tina L, Ngwa Verra M, Benian Guy M
Department of Pathology, Emory University, Atlanta, GA 30322.
Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
Mol Biol Cell. 2016 May 15;27(10):1606-20. doi: 10.1091/mbc.E15-09-0675. Epub 2016 Mar 23.
UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89's SH3 domain and residues 294-376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is α-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue," which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.
UNC-89是一种位于秀丽隐杆线虫肌肉肌节M线的巨大多肽。其人类同源物是 obscurin。为了了解UNC-89是如何定位和发挥功能的，我们一直在鉴定其结合伴侣。筛选酵母双杂交文库发现UNC-89与副肌球蛋白相互作用。副肌球蛋白是一种无脊椎动物特有的卷曲螺旋二聚体蛋白，与肌球蛋白重链的杆状部分同源，存在于粗肌丝核心中。这种相互作用至少需要UNC-89的SH3结构域和副肌球蛋白的294 - 376位残基，解离常数约为1.1 μM。在缺乏SH3结构域的unc-89功能丧失突变体中，副肌球蛋白会聚集。当SH3结构域过表达时，副肌球蛋白会定位错误。SH3结构域通常与富含脯氨酸的共有序列相互作用，但副肌球蛋白与UNC-89的SH3相互作用的区域是α螺旋，且缺乏脯氨酸。UNC-89的SH3的同源建模表明了可能负责这种相互作用的结构特征。副肌球蛋白的SH3结合区域包含一个“跳跃残基”，它可能会使卷曲螺旋局部解旋，也许有助于结合特异性。
